Immunological detection of N-formylkynurenine in oxidized proteins

Laboratory of Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Free Radical Biology and Medicine (Impact Factor: 5.74). 06/2009; 46(9):1260-6. DOI: 10.1016/j.freeradbiomed.2009.01.020
Source: PubMed


Reactions of tryptophan residues in proteins with radical and other oxidative species frequently lead to cleavage of the indole ring, modifying tryptophan residues into N-formylkynurenine (NFK) and kynurenine. Tryptophan modification has been detected in physiologically important proteins and has been associated with a number of human disease conditions. Modified residues have been identified through various combinations of proteomic analyses, tryptic digestion, HPLC, and mass spectrometry. Here we present a novel, immunological approach using polyclonal antiserum for detection of NFK. The specificity of our antiserum is confirmed using photooxidation and radical-mediated oxidation of proteins with and without tryptophan residues. The sensitivity of our antiserum is validated through detection of NFK in photooxidized myoglobin (two tryptophan residues) and in carbonate radical-oxidized human SOD1, which contains a single tryptophan residue. Analysis of photooxidized milk also shows that our antiserum can detect NFK residues in a mixture of proteins. Results from mass spectrometric analysis of photooxidized myoglobin samples corroborate the immunological data, detecting an increase in NFK content as the extent of photooxidation increases.

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    • "The previously described anti-NFK antiserum [26] [27] [28] was developed in our laboratory, and mouse monoclonal anti-αA and anti-αB crystallin and goat polyclonal anti-actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The (goat) anti-mouse IRdye 800 used in Western analysis was from Li-Cor Biosciences (Lincoln, NE, USA) and the DAPI and all secondary antibodies used in the confocal experiments were purchased from Life Technologies ( "
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    • "Post-translational oxidation of Trp to form N-formylkynurenine (NFK) (Figure 1A) plays a role in oxidative stress responses in some proteins (see for example [13]). NFK has been identified in mitochondrial ATP synthase [14], spinach LHCII [15], milk proteins [16], skeletal muscle proteins [17], apolipoprotein B-100 [13], and Methylococcus capsulatus-secreted MopE protein [18]. "
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    • "The highest complexity of oxidation products was observed for W432 in both HisRS* and recHisRS* (Fig. S1). 1 The oxidation of tryptophan is known to consist of 3 sequential steps resulting in the formation of oxidized tryptophan, N′-formylkunurenine, and kynurenine, respectively (Fig. S2) 1 (Taylor et al. 2003). The presence of (formyl)kynurenine has been described in mitochondrial proteins, but has so far never been linked to an autoantigen or to autoimmunity (Ehrenshaft et al. 2009). The oxidative HisRS modifications might generate epitopes recognized by the immune system. "
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